Field of the Invention (Technical Field)
The present invention relates to synthetic growth factor modulator compositions, particularly modulators of the Bone Morphogenic Protein (BMP) family. Compositions of the present invention are of the formulas disclosed herein with a single or dual chain peptide sequence having specific binding affinity to a BMP-2 receptor, a linker, optionally a hydrophobic linker, and a non-growth factor heparin-binding sequence, and methods of use of synthetic growth factor modulators.
Background Art
Note that the following discussion refers to a number of publications by author(s) and year of publication, and that due to recent publication dates certain publications are not to be considered as prior art vis-à-vis the present invention. Discussion of such publications herein is given for more complete background and is not to be construed as an admission that such publications are prior art for patentability determination purposes.
Bone Morphogenic Proteins (BMPs) are a group of proteins involved in the development of a wide range of organs and tissues from embryonic through adult stages, (Wozney J M 2002, Spine 27(16 Suppl 1):S2-8). BMPs also play important roles in tissue repair and remodeling processes following injuries. Certain BMPs induce ectopic bone formation and enhance healing of critical-sized segmental bone defects in animal models. Clinical studies show that recombinant human BMPs (rhBMPs) are safe and effective alternatives to autologous bone grafting. rhBMP-2 and rhBMP-7 are approved for human use in spinal fusion and recalcitrant long-bone nonunions, respectively. (Kleeman et al. 2001, Spine 26(24):2751-6. Burkus et al. 2002. Spine 27(21):2396-408. McKay et al. 2002, Spine 27(16 suppl 1):S66-85. Poynton et al. 2002. Spine 27(16 suppl 1):S40-8.)
The effectiveness of rhBMP-2 seems to heavily depend on the dose. Significantly higher-than-physiological doses are required for therapeutic effect in vivo. For example, levels in the neighborhood of 1 mg/mL of rhBMP-2 are used in spinal fusion cages (up to 8 mg/cage), an amount three orders of magnitude higher than what is typically found endogenously. (McKay et al. 2002, Spine 27(16 suppl 1):S66-85.) Administration of such a high dose of recombinant protein is not only costly, but may also be associated with adverse effects such as bony overgrowth and immunological reactions. Therefore, the development of positive modulators of BMP-2 to enhance BMP activities is of clinical significance.
BMP-2 signaling involves two types of transmembrane serine/threonine kinase receptors, namely type I (BRI) and type II (BRII). (Hoodless et al 1996, Cell 85(4):489-500. Kawabata et al. 1995, J Biol Chem 270(10):5625-30. Nohno et al. 1995, J Biol Chem 270(38):22522-6. Rosenzweig et al. 1995, Proc Natl Acad Sci USA 92(17):7632-6.) An active ligand/receptor complex consists of BMP-2, BRI, and BRII in a 2:2:2 ratio. (Reddi A H 2001, J Bone Joint Surg Am 83-A Suppl 1 (Pt 1):S1-6) Both type I and type II receptors are required for BMP-2 to exert its biological functions. Upon BMP-2 binding. BRI kinase is activated as a result of phosphorylation by BRII. BRII would not bind to BMP-2 without the presence of BRI and the complex of BMP-2 and BRII is not capable of initiating signaling in the absence of BRI. The serine/threonine kinase in the BRI receptor is believed to be responsible for the phosphorylation of Smad1, Smad5, and Smad8, which in turn assemble into heteromeric complexes with Smad4 and translocate into the nucleus to regulate transcription of target genes. (Massague et al. 2000, Genes Dev 14(6):627-44. Attisano et al. 2000. Curr Opin Cell Biol 12(2):235-43.) In addition, the activated receptor complexes can activate the p38 MAP kinase pathway independent of the Smad pathway. (Iwasaki et al. 1999, J Biol Chem 274(37):26503-10. Miyazono K 2000. J Cell Sci 113(Pt 7):1101-9.) Currently there are thought to be two modes for BMP-2 to initiate signaling. Gilboa and colleagues showed that multiple BMP receptor oligomers are present at the cell surface prior to ligand binding. (Gilboa et al 2000, Mol Biol Cell 11(3):1023-35.) Nohe and colleagues then showed that the pre-formed receptor complexes are responsible for the BMP-2 induced Smad pathway activation, and BMP-2-induced receptor complexes initiate the p38 kinase pathway. (Nohe et al. 2002, J Biol Chem 277(7):5330-8.)
Some efforts have been made to generate heparin-binding growth factor analogs. For example, natural platelet-derived growth factors (PDGF) occur as an A chain and a B chain arranged in head-to-head (AA or BB) homodimers, or (AB or BA) heterodimers. Thus, U.S. Pat. No. 6,350,731 to Jehanli et al. discloses PDGF analogs in which two synthetic PDGF receptor-binding domains are covalently linked through a polyglycine or an N-(4-carboxy-cyclohexylmethyl)-maleimide (SMCC) chain to mimic the natural active polypeptide dimer.
U.S. Pat. No. 6,235,716 to Ben-Sasson discloses analogs of angiogenic factors. The analogs are branched multivalent ligands that include two or more angiogenic homology regions connected by a multilinker backbone.
U.S. Pat. No. 5,770,704 (the '704 patent) to Godowski discloses conjugates for activating receptor tyrosine kinases, cytokine receptors and members of the nerve growth factor receptor superfamily. The conjugates include at least two ligands capable of binding to the cognate receptor, so that the binding of the respective ligands induces oligomerization of these receptors. The ligands disclosed in the '704 patent are linked by covalent attachment to various non-proteinaceous polymers, particularly hydrophilic polymers, such as polyvinylalcohol and polyvinylpyrrolidone, and the polyvinylalkene ethers, including polyethylene glycol and polypropylene glycol. The ligands include hepatocyte growth factor (HGF) peptide variants that each bind HGF receptor, thereby causing receptor dimerization and activation of the biological activity of the HGF receptor dimer.
U.S. Pat. No. 6,284,503 (the '503 patent) to Caldwell et al. discloses a composition and method for regulating the adhesion of cells and biomolecules to hydrophobic surfaces and hydrophobic coated surfaces for cell adhesion, cell growth, cell sorting and biological assays. The composition is a biomolecule conjugated to a reactive end group activated polymer. The end group activated polymer includes a block copolymer surfactant backbone and an activation or reactive group. The block copolymer may be any surfactant having a hydrophobic region capable of adsorbing onto a hydrophobic surface, and a hydrophilic region which extends away from the surface when the hydrophobic region is adsorbed onto the hydrophobic surface. The '503 patent discloses that the biomolecules that may be conjugated to the end group activated polymer include natural or recombinant growth factors, such as PDGF, EGF, TGFα, TGFβ, NGF, IGF-I, IGF-II, GH and GHRF, as well as multi-CSF(II-3), GM-CSF, G-CSF, and M-CSF.
Other workers have described compositions that include homologs and analogs of fibroblast growth factors (FGFs). See for example U.S. Pat. No. 5,679,673 to Lappi and Baird; U.S. Pat. No. 5,989,866 to Deisher et al. and U.S. Pat. No. 6,294,359 to Fiddes et al. These disclosures relate to FGF homologs or analogs that are either conjugated to a toxic moiety and are targeted to the FGF receptor-bearing cells; or are homologs or analogs that modulate the biological pathways through the signal transduced by the FGF receptor upon binding by the FGF homolog or analog.
A series of patent applications to Kochendoerfer et al disclose polymer-modified proteins, including synthetic chemokines and erythropoiesis stimulating proteins. See, for example, International Publications WO 02/04105, WO 02/19963 and WO 02/20033. These include chemically ligated peptide segments of a polypeptide chain of a synthetic erythropoiesis protein, such that a polypeptide chain results, with a water soluble polymer attached at one or more glycosylation sites on the protein. These applications also disclose synthetic chemokines, which are also polymer modified, and are asserted to be antagonists. However, heparin-binding domains are not disclosed. Other erythropoietin mimetics are known, such as those disclosed in U.S. Pat. No. 5,773,569 and 5.830.851 to Wrighton et al.
International Publication WO 00/18921 to Ballinger and Kavanaugh discloses a composition consisting of fusion proteins having FGF receptor affinity linked to an “oligomerization domain”, either directly or through a linking group. The oligomerization domain ranges in length from about 20 to 300 residues, and includes constructs such as transcription factors, Fc portions of IgG, leucine zippers and the like. The oligomerization domains disclosed are homodimeric domains, wherein a single FGF receptor affinity fusion protein is linked to a single domain, such as a leucine zipper, which in turn is linked to a similar molecule by means of cysteine residues at both the amino and carboxy termini of the leucine zippers, such that two parallel leucine zippers, each with a single FGF receptor affinity fusion protein, are cross-linked by means of disulfide bonds. It is also disclosed that fusion proteins may include a heparin binding domain, such as the use of jun as a multimerization domain, which is asserted to be a heparin binding domain. Thus the compositions disclosed by Ballinger and Kavanaugh are all composed of a single receptor-binding sequence covalently attached to an oligomerization domain, whereby two or more similar oligomerization domains, each with a single receptor-binding sequence, are conjoined by means of either an association provided by the oligomerization domain, or alternatively, are chemically cross-linked to provide for the covalent bonding of the individual components.
The above described homologs, analogs, conjugates or ligands each include a receptor-binding domain. However, none of the disclosed compounds or compositions further include both a linker, providing for the linking of two receptor-binding domains to a dipeptide sequence, and further providing a single non-signaling peptide containing a heparin-binding domain. Moreover, none of these or other known heparin-binding growth factor analogs provide the advantages described herein below. Further, the prior art does not disclose modulators which, through a synergistic effect, increase or enhance the efficacy of a naturally occurring growth factor, such as BMP-2.